Reconstitution of human lymphocytes following ex vivo expansion of human umbilical cord blood CD34+ cells and transplantation in Rag2-/- gamma c-/- mice model.
BACKGROUND
Due to ethical issues, in vivo studies of the human immune system have been difficult. Thus, small-animal xenotransplantation models have been employed, although they scarcely sustain a human immune response. In this study, we compared human cell repopulation tendencies and functionality in Rag2-/- gamma c-/- mice following various ex vivo expanded human hematopoietic stem cells (HSCs).


METHODS
Human umbilical cord blood (UCB) CD34+ cells were cultured for 7 days with a cytokine combination of stem cell factor, Flk2/Flt3 ligand, and thrombopoietin, with absence or presence of rhIL-3, then transplanted into Rag2-/- gamma c-/- mice. Reconstituted human lymphocytes were analyzed based on the expression of CD45 as well as CD3, CD19, and CD56 in peripheral blood (PB) until 16 weeks after transplantation. BrdU assay and functional analysis of reconstituted human lymphocytes used PHA- or rhIL-2-stimulated splenocytes and bone marrow cells from recipient mice.


RESULTS
The percentage of human CD45dim cells, not CD45bright cells, in PB of mice transplanted with cultured HSCs with rhIL-3 was much higher than in the group without rhIL-3 (approximately 2.5-fold at week 10 posttransplantation). The humanized mice showed systemic repopulation with a comprehensive array of human lymphohematopoietic cells, including T, B, natural killer (NK) cells, and even dendritic cells. However, the expression level was also dim. The number of CD3+ T cells and CD56+ NK cells was especially increased in the presence of rhIL-3. In addition, after in vitro restimulation proliferation assays and NK activity of interferon-gamma secretion showed greater effects in the presence of rhIL-3.


CONCLUSIONS
These data suggested that the development of a diverse repopulation of human lymphocytes was possible in Rag2-/- gamma c-/- mice after transplantation of cultured UCB CD34+ HSCs with interleukin-3.
Choi
B K
BK
Transplantation Research Center, Samsung Biomedical Research Institute, Seoul, South Korea.
Joo
S Y
SY
Moon
C
C
Park
K S
KS
Kim
S H
SH
Park
J B
JB
Jung
G O
GO
Choi
G S
GS
Kwon
C H
CH
Chun
J M
JM
Joh
J W
JW
Lee
S K
SK
Kim
S J
SJ
D000818:Animals; D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000276:immunology*; D018929:Cell Culture Techniques; D017690:Cell Transplantation / Q000379:methods*; D004268:DNA-Binding Proteins / Q000172:deficiency*; D005312:Fetal Blood / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007231:Infant, Newborn; D007377:Interleukin-3 / Q000494:pharmacology; D017493:Leukocyte Common Antigens / Q000097:blood; D051379:Mice; D018345:Mice, Knockout; D011994:Recombinant Proteins / Q000494:pharmacology; D014183:Transplantation, Heterologous / Q000379:methods*; D014471:Umbilical Veins
D015703:Antigens, CD; D018952:Antigens, CD34; D004268:DNA-Binding Proteins; D007377:Interleukin-3; C499308:Rag2 protein, mouse; D011994:Recombinant Proteins; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human